Eukaryotic chromosomes terminate in structures called telomeres, which contain tandem sequence repeats. With each cell replication cycle, telomeres become progressively shorter due to the inability of the DNA replication machinery to completely synthesize the opposing strand. Shortened telomeres induce replicative senescence, which is avoided in rapidly proliferating cells by the activation of the reverse transcriptase telomerase. Telomerase is a ribonucleoprotein that adds tandem repeats to the ends of telomeres to maintain length, and is both positively and negatively regulated in vivo. Dysregulation of telomerase leads to several human diseases, including cancer, aplastic anemia, dyskeratosis congenita, and idiopathic pulmonary fibrosis. Several models of the molecular mechanisms of telomerase regulation have been developed based in vivo experiments. The proposed studies will use standard biochemical and biophysical techniques to evaluate the structures of the telomere-associated proteins Cdc13 and Est1, which will be used to develop a novel in vitro system of reconstituted yeast telomerase. This system will be used to rigorously study the mechanisms of telomerase activity and regulation that have been genetically identified in vivo. Yeast model systems have previously provided considerable insight into human telomere maintenance, and a wealth of yeast genetic data is available to rigorously evaluate the function of individual proteins in the system. In Aim 1, we will determine the structure of the telomerase-recruitment domain of the telomere-binding protein Cdc13, and assay the role of this domain in DNA binding by full-length Cdc13. Aim 2 will characterize the biochemical activities of the telomerase regulatory subunit Est1, which will be recombinantly produced in insect cells to generate large yields of a high-quality reagent that is critically needed in the field. Aim 3 will test the central element of the recruitment model of telomerase activation by determining if a direct physical interaction between Cdc13 and Est1 exists, and then evaluating interactions between genetically identified mutants of both proteins. Aim 4 will reconstitute the entire telomerase complex in vitro for the first time, using recombinant reagents that have been extensively characterized. Activity assays will be performed using this novel system to fully test the Cdc13/Est1 recruitment model of telomerase activation. These in vitro studies of the molecular mechanisms of yeast telomerase activity will increase our general understanding of telomerase activity and regulation, and will specifically provide a framework for the dissection of the molecular mechanisms of human telomere maintenance that are disrupted in disease. PUBLIC HEALTH RELEVANCE: The ends of linear chromosomes comprise specialized structures, called telomeres, that have essential roles in aging, genomic stability, and cancer. The length of the telomere is a marker of cellular aging, and sufficiently shortened telomeres arrest cell growth. Nearly all human cancers evade that checkpoint by activating the enzyme telomerase, which restores telomere length through the addition of DNA sequence. However, insufficient telomerase activity can also cause chronic fatal diseases such as aplastic anemia. The proper maintenance of telomeres and regulation of telomerase are therefore essential for human health.